The microbial diversity of the planet is primarily represented by uncultivated species that are not amenable to growth under laboratory conditions. Obligate anaerobes are a particular example of microbes that are not amenable to cultivation using standard laboratory techniques as these cells are either completely or nearly completely intolerant to oxygen exposure. Obligate anaerobic microorganisms employ anaerobic respiration that may include fermentative routes to obtain energy. Some of these pathways include propionic acid fermentation, butyric acid fermentation, mixed acid fermentation, butanediol fermentation, Stickland fermentation, acetogenesis or methanogenesis. There is a great deal of metabolic diversity exhibited by anaerobic microorganisms, some of which may lead to fermentation products such as wine. More recently, there is an increased interest in the production of alcohols such as ethanol and butanol derived from plant materials to produce biofuels to substitute petrochemical-derived combustibles as vehicle propellant.
Some of these previously uncultivated anaerobes are thought to mediate processes in the environment linked to global carbon and energy fluxes, including the production methane, biodiesel, hydrogen and alcohols from carbon sources. Methane extracted from coal deposits, known as coal bed methane, from which commercial methane gas is produced, is thought to be the product (in whole or in part) of such anaerobic organisms. New methods for isolating and cultivating novel anaerobic microorganisms are desirable, since such organisms have a variety of useful applications. For example, the ability to cultivate novel anaerobic species participating in the methane production at coal bed sites will allow for increased methane production.
The isolation and cultivation of anaerobic microorganisms has been limited to date by the lack of available equipment and culture vessels providing anoxic atmospheres on which oxygen sensitive cells divide and cultures may be established. High speed cell sorters, such as flow cytometers, have been used for the selection and downstream cultivation of aerobic microorganisms, and in some cases instrument modifications have been made to allow cell sorting under controlled atmospheres, including anaerobic gas mixtures. However the complete operation of a cell sorting device in a contained environment has never been demonstrated. This is thought to be primarily due to their relatively large dimensions and complex ancillary infrastructure required for the operation of typical cell sorting devices, such as water cooling systems, pressurized air, and very sensitive 3D laser alignment.
Conventional gel microdroplet (or “GMD”) encapsulation and flow cytometry have been used for studying aerobic bacteria (Nir et al. 1990 Appl. Environ. Microbiol. 56:2870-2875; Katsuragi et al., 2000 J. Microbiol. Meth. 42:81-86; Manome et al. 2001 FEMS Microbiol. Lett. 197: 29-33). Zengler et. al. (2002 PNAS, 99:15681-15686) used GMDs for growth of novel aerobic bacteria from the environment demonstrating the ability of the GMD technique combined with flow cytometry to cultivate previously uncultured microorganisms. The authors also grew one strain of the strict anaerobe Methanococcus thermolithotrophicus from the American Type Culture Collection in GMDs, but did not demonstrate the ability to sort this anaerobically.
One limitation to sorting cells anaerobically is the inability to perform cell sorting under anoxic conditions. Dissolved oxygen present in the fluidics and during sample injection exposes cells to oxygen levels not permissible for anaerobic growth. Establishment of anaerobic cultures using aerobic procedures, such as those found in existing high speed cell sorting, is hindered by the cellular damage produced by oxygen upon brief exposure time. Thus, the vast majority of strict anaerobic cells cannot be sorted using existing technology. As a consequence, work to date conducting aerobic or partially anaerobic cell sorting of anaerobic cells has resulted in an incomplete cultivation of the indigenous populations.
An example of a partially enclosed device and method for processing cells is described in U.S. Pat. No. 6,780,377 (hereinafter “the '377 patent”). The '377 patent provides an environmental containment system fitted to a DAKO cell sorter to sort cells under a specific atmosphere including those suitable for the cultivation of strict anaerobic microorganisms. A major limitation of this environmental containment system is that the sample cells are exposed to oxygen at multiple points during processing. First, to sort obligate anaerobes, the sample needs to be prepared and handled under anaerobic conditions. This implies at a minimum that the transportation of the sample between an anaerobic chamber used to prepare the sample and the cell sorting system needs to be performed in an oxygen-free environment. This point is not discussed in the '377 patent. Second, introduction of the sample into the cell sorting device must also take place in an oxygen-free environment. As such, the sample port of the cell sorter needs to be free of oxygen. The '377 patent does not considered the sample injection port as part of the anaerobic environment. As a result, a sample processed using the invention described in that patent is exposed to oxygen from the air prior to cytometry and sorting. Third, the fluidics driving the laminar flow and producing the envelope and droplets used during cell sorting need to be oxygen free. The necessity of maintain not only an anaerobic gas but the use of anaerobic fluids, that is, through sparging anaerobic gas into a suitable gas impermeable container as part of the process is not addressed and it is only mentioned the potential for “adjustably controlling the environment surrounding the process(es) of fluid stream generation, droplet formation, droplet separation and droplet collection.” In practical terms, the use of fluids with low amounts of oxygen (few parts per million) can be toxic for anaerobic microorganisms and the fluids used to drive the sample and droplet formation need to absolutely oxygen free. In view of these systemic limitations, it is clear that the '377 patent does not teach a completely anoxic system.
What is needed is a cell sorting system, the components of which are completely self-contained, which protects organisms from exposure to oxygen prior to, during, and after the cell sorting process.